Induced encystment improves resistance to preservation and storage of Acanthamoeba castellanii

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1 Induced encystment improves resistance to preservation and storage of Acanthamoeba castellanii 141 S. J. CAMPBELL 1,P.R.INGRAM 2, C. W. ROBERTS 2 and F. L. HENRIQUEZ 1 * 1 School of Engineering and Science, University of the West of Scotland, Paisley PA1 2BE, Scotland 2 Strathclyde Institute of Pharmacy and Biomedical Sciences, University of Strathclyde, Glasgow G4 NR, Scotland (Received 9 June 8; revised 4 July and 4 August 8; accepted 12 August 8) SUMMARY Several conditions that allow the preservation, storage and rapid, efficient recovery of viable Acanthamoeba castellanii organisms were investigated. The viability of trophozoites (as determined by time to confluence) significantly declined over a period of 12 months when stored at x7 xc using dimethyl sulfoxide (DMSO; or %) as cryopreservant. As A. castellanii are naturally capable of encystment, studies were undertaken to determine whether induced encystment might improve the viability of organisms under a number of storage conditions. A. castellanii cysts stored in the presence of Mg 2+ at 4 xc remained viable over the study period, although time to confluence was increased from approximately 8 days to approximately 24 days over the 12-month period. Storage of cysts at x7 xc with DMSO ( or %) or 4% glycerol, but not 8% glycerol as cryopreservants increased their viability over the 12-month study period compared with those stored at room temperature. Continued presence of Mg 2+ in medium during storage had no adverse effects and generally improved recovery of viable organisms. The present study demonstrates that A. castellanii can be stored as a non-multiplicative form inexpensively, without a need for cryopreservation, for at least 12 months, but viability is increased by storage at x7 xc. Key words: Acanthamoeba, preservation, storage, cyst, trophozoite. INTRODUCTION Acanthamoeba species are normally free-living protozoa, their transmission and spread does not depend on a host and they are found almost ubiquitously in the environment (reviewed by Marciano-Cabral and Cabral, 3). Several species are pathogenic, causing granulomatous amoebic encephalitis (GAE) and cutaneous acanthamoebiasis in immunocompromised or immunosuppressed hosts, and amoebic keratitis (AK) in immunocompetent individuals (reviewed by Marciano-Cabral and Cabral, 3; Schuster and Visvesvara, 4). As the importance of this pathogen is recognized there has been an increase in the number of studies, particularly those investigating new antimicrobials, that may improve treatment (Schuster and Visvesvara, 1998; Mattana et al. 4; McBride et al. 7; Henriquez et al. 8). The ability to preserve protozoa in a non-actively dividing state has a number of advantages. Firstly, it reduces the material and labour costs of continued culture and passage, secondly it reduces the chance of selection for culture-adapted organisms observed during continued tissue culture and it also allows the * Corresponding author: School of Engineering and Science, University of the West of Scotland, Paisley PA1 2BE, Scotland. Tel: Fax: fiona.henriquez@uws.ac.uk possibility to maintain more than one isolate in a single location. Furthermore, it may facilitate transport of isolates without regard to time. The cryopreservation of Acanthamoeba and other free-living amoebae has been previously described (Seo et al. 1992; John et al. 1994; John and John, 1996; Alejandre-Aguilar et al. 1998; Gonzalez-Robles et al. 1). The slow cooling (overall. 7 xc/min) of freeliving amoeba trophozoites, Acanthamoeba culbertsoni, A. polyphaga, Naegleria fowleri and N. gruberi, with 7. % dimethylsulfoxide (DMSO) or 7. % glycerol produced 2 39% viable trophozoites after 6 days in liquid nitrogen (Seo et al. 1992). John et al. (1994) reported that recovery of A. castellanii trophozoites decreases with longer storage times at x7 xc with 12% DMSO, % heat-inactivated bovine calf serum and 4% glucose with the greatest reduction during the first year of storage (John and John, 1996). Alejandre-Aguilar et al. (1998) found that the viability of A. castellanii trophozoites increased when stored with % DMSO in liquid nitrogen if an equilibrium temperature of 4 xc was used. As we found that our ability to recover viable trophozoites from cryopreservation reduced with time we sought an alternative method. Acanthamoeba exist as active trophozoites or dormant cysts. Encystment occurs when the organism is exposed to adverse environmental conditions such as absence of nutrients, biocides, fluctuations in temperature and Parasitology (8), 13, f 8 Cambridge University Press doi:.17/s Printed in the United Kingdom

2 S. J. Campbell and others 142 % DMSO % DMSO Fig. 1. The average time (days) until confluence for Acanthamoeba castellanii trophozoites stored in RM with % or % DMSO at x7 xc. The length of time to reach confluence increased the longer time spent in storage for all samples from 6. to 16 days. The data represent the mean of 2 replicate experiments. ph (reviewed by Schuster, 2). Encystment can be induced in the laboratory by the addition of MgCl 2 to the medium (reviewed by Schuster, 2). We hypothesized that encystment of Acanthamoeba may increase their viability during cryopreservation or eliminate the need for it completely. Herein, a number of different storage conditions for A. castellanii trophozoites and cysts are compared. The effectiveness of each condition was determined via microscopical observations of the time required to reach confluence. MATERIALS AND METHODS Maintenance of A. castellanii trophozoites Acanthamoeba castellanii (Neff strain) was kindly donated by Keith Vickerman (Glasgow, UK). Trophozoites were routinely grown in RM containing 2% mycological peptone (Sigma, Poole, UK) and. 9% maltose (Sigma, Poole, UK), supplemented with 1 mg penicillin/streptomycin and 2. mg/ml amphotericin B (Sigma, Poole, UK) as previously described (McBride et al. ). They were incubated until confluent at room temperature in 7-cm 2 tissue-culture flasks, followed by either subculture or harvest by mechanical detachment. Preparation of cysts The RM was removed from confluent 7-cm 3 tissueculture flasks and replaced by ml of encystment medium (EM) ( mm Tris-HCl, ph8. 8, mm KCl, 8 mm MgSO 4,. 4mMCaCl 2,1mMNaHCO 3 all reagents from Sigma) (Neff et al. 1964) and monitored until cysts had formed (2 3 days). The cysts were then collected by centrifugation at g for min resuspended in (EM) and counted using a haemocytometer. Cysts were adjusted to 3r /ml, added to microcentrifuge tubes (ThermoElectron, UK) and centrifuged at g for min. The EM was then removed and the cysts were resuspended in 1 ml of the appropriate medium for storage: (1) EM or RM with (a) % DMSO, (b) % DMSO, (c) 2. % Fig. 2. The average time (days) until confluence for Acanthamoeba castellanii cysts stored in EM at 4 xc. The length of time to reach confluence increased the longer time spent in storage from 8. to 24 days. The data represent the mean of 2 replicate experiments. DMSO, (d) 8% glycerol, (e) 4% glycerol and (f) % glycerol or (2) EM alone. EM and RM suspensions with glycerol or DMSO were then transferred to a cryopreservation tube and stored at x7 xc and EM alone was placed at 4 xc for 1, 3, 6 and 12 months. Preparation of trophozoites Trophozoites were collected from newly confluent cultures by mechanical disruption followed by centrifugation at g for min and resuspended in RM. Then 3r trophozoites in 1 ml of suspension were added to microcentrifuge tubes and centrifuged at g for min. The RM was then removed and the trophozoites were resuspended in RM, counted using a haemocytometer and adjusted to 3r /ml in the appropriate medium for storage: RM with (a) % DMSO, (b) % DMSO, (c) 2. % DMSO, (d) 8% glycerol, (e) 4% glycerol and (f) % glycerol. The trophozoite suspensions were then transferred to a cryopreservation tube (Nunc, Denmark) and stored at x7 xc for 1, 3, 6 and 12 months. Recovery of Acanthamoeba After the appropriate length of time in storage (1, 3, 6 and 12 months) each cryovial was thawed slowly at

3 Improved preservation and storage of Acanthamoeba castellanii 143 A % DMSO % DMSO B 8% glycerol 4% glycerol Fig. 3. The average time (days) until confluence for Acanthamoeba castellanii cysts stored at x7 xc in RM with (A) % or % DMSO and (B) 8% or 4% glycerol. (A) The length of time to reach confluence increased for cysts stored with RM and % DMSO the longer time spent in storage from 8. to days whereas cysts stored with % DMSO varied from 8. to 14 days over the 12-month period. (B) The length of time until confluence remained constant for cysts stored in 4% glycerol and RM ( 12. days) whereas cysts stored with 8% glycerol increased after from 11 to 24 days. The data represent the mean of 2 replicate experiments. room temperature and then transferred to a microcentrifuge tube. Cells were collected by a -min centrifugation at 13 rpm and 4 xc, resuspended in ml of RM the transferred to cm 2 flasks. All samples were monitored every 24 h until the flasks containing either trophozoites or cysts, which revert back to trophozoites in RM, reached % confluency. RESULTS A. castellanii trophozoites and cysts were stored under various conditions to determine their optimal long-term storage method. At 1, 3, 6 and 12 months after being placed in storage, A. castellanii were placed in RM until % confluency was reached (Figs 1 4). This demonstrated their viability. Both Acanthamoeba trophozoites and cysts remained viable in all storage media, with the exception of those stored in media containing % and % glycerol (results not shown). The length of time to reach % confluency increased for A. castellanii trophozoites stored in RM with % or % DMSO at x7 xc from 6. to 16 days as the length of time to storage increased (Fig. 1). The time to % confluency also increased for A. castellanii cysts stored in EM at 4 xcfrom8. to 24 days as the time in storage increased from 1 to 12 months (Fig. 2). The length of time to % confluency increased for A. castellanii cysts stored in RM and % DMSO with length of time spent in storage (from 8. to days). However, at a DMSO concentration of % the length of time to confluence varied throughout the entire study, with an increase from 1 month to 3 months (8. to12. days), then decreasing after 6 months (8. days) and finally increasing after 12 months (14 days) (Fig. 3). For cysts stored in 4% glycerol and RM the length of time to confluence remained constant ( 12. days), whereas for cysts stored in 8% glycerol the time required to reach trophozoite confluence increased (from 11 to 24) over the 12-month study period (Fig. 3). The average time until confluence for A. castellanii cysts stored at x7 xc in EM and in % or % DMSO varied from 9 to 13 days over the 12-month period (Fig. 4). However, for A. castellanii cysts stored in EM with 8% glycerol the time to confluence increased over the study period from 13 to 24 days and cysts stored in 4% glycerol remained constant at 12. days (Fig. 4). DISCUSSION Cryopreservation of Acanthamoeba species has been previously described in a number of studies (Seo et al. 1992; John et al. 1994; John and John, 1996; Alejandre-Aguilar et al. 1998; Gonzalez-Robles et al. 1). A solution containing % DMSO had previously been shown to produce the highest number of viable A. castellanii trophozoites after storage in liquid nitrogen for up to 2 days (Alejandre-Aguilar et al. 1998). Other studies have also demonstrated

4 S. J. Campbell and others 144 A % DMSO % DMSO B 8% glycerol 4% glycerol Fig. 4. The average time (days) until confluence for Acanthamoeba castellanii cysts stored at x7 xc in EM with (A) % or % DMSO and (B) 8% or 4% glycerol. (A) The length of time for cysts in % or % DMSO varied from 9 to 13 days over the 12-month period. (B) Cysts stored in EM with 8% glycerol took a lot longer to reach confluence (13 24 days) than cysts stored in 4% glycerol ( 12. days). The data represent the mean of 2 replicate experiments. that the viability of A. castellanii trophozoites decreases during the first year of storage in the presence of 12% DMSO at x7 xc (John et al. 1994). The studies undertaken herein, to investigate alternative means of storage, were instigated by similar anecdotal observations in our laboratory. Acanthamoeba transform into cystic stages under adverse conditions and are resistant to biocides, chlorination and antibiotics. Cysts have been shown to give rise to viable trophozoites after 24 years in storage at 4 xc (Mazur et al. 199; Marciano-Cabral and Cabral, 3), but their viability has not previously been assessed relative to cryopreserved trophozoites. The results of the present study confirm that viability as determined by time to confluence is reduced in stabilates stored with DMSO or glycerol as cryopreservant following only 1 month of storage at x7 xc. However, cysts in EM stored at 4 xc remain viable for in excess of 1 year although time to reach confluence is increased with time in storage, up to 12 months. Specifically, time to reach confluence increased from 8. to 24 days over this time-period. Viability could be markedly improved by storage at x7 xc in the presence of either DMSO or glycerol as cryopreservant. Notably, those stored in 4% glycerol remained relatively constant throughout the whole time-period, reaching confluence at 8. days after the first month of storage and between and 12. days for time-periods up to and including 1 year. Similar results were obtained using % and % DMSO as cryopreservant. The presence of Mg 2+ used to induce encystment was not necessary in the cryo-medium, but did not have an adverse affect on amoeba viability. Overall these studies demonstrate that A. castellanii are best stored as cyst stages rather than as trophozoites. In this state they can be easily and inexpensively preserved for at least 12 months without the need for cryopreservation. However, preservation can be improved when stored x7 xc with cryo-preservant and this refinement is recommended for long-term storage where facilities exist. REFERENCES Alejandre-Aguilar, R., Calvo-Mendez, L., Nogueda-Torres, B. and de la Jara-Alcocer, F. (1998). Maintenance of Acanthamoeba culbertsoni by cryopreservation. The Journal of Parasitology 84, Gonzalez-Robles, A., Flores-Langarica, A., Omana-Molina, M. and Shibayama, M. (1). Acanthamoeba castellanii: ultrastructure of trophozoites using fast free fixation. Journal of Electron Microscopy, Henriquez, F. L., Ingram, P. R., Muench, S. P., Rice, D. W. and Roberts, C. W. (8). Molecular basis for resistance of Acanthamoeba tubulins to all major classes of antitubulin compounds. Antimicrobial Agents and Chemotherapy 2, John, D. T., Eddy, P. L. and John, R. A. (1994). Cryopreservation of pathogenic free-living amebae. Folia Parasitologica 41, John, D. T. and John, R. A. (1996). Viability of pathogenic Acanthamoeba and Naegleria and virulence

5 Improved preservation and storage of Acanthamoeba castellanii 14 of N. fowleri during long-term cryopreservation. Folia Parasitologica 43, Marciano-Cabral, F. and Cabral, G. (3). Acanthamoeba spp. as agents of disease in humans. Clinical Microbiology Reviews 16, Mattana, A., Biancu, G., Alberti, L., Accardo, A., Delogu, G., Fiori P. L. and Cappuccinelli, P. (4). In vitro evaluation of the effectiveness of the macrolide rokitamycin and chlorpromazine against Acanthamoeba castellanii. Antimicrobial Agents and Chemotherapy 48, Mazur, T., Hadaś, E. and Iwanicka, I. (199). The duration of the cyst stage and the viability and virulence of Acanthamoeba isolates. Tropical Medicine and Parasitology 46, 6 8. McBride, J., Ingram, P. I., Henriquez, F. L. and Roberts, C. W. (). Development of a colourimetric microtitre plate assay for assessment of anti-microbial agents against Acanthamoeba. Journal of Clinical Microbiology 43, McBride, J., Mullen, A. B., Carter, K. C. and Roberts, C. W. (7). Differential cytotoxicity of phospholipid analogues to pathogenic Acanthamoeba species and mammalian cells. Journal of Antimicrobial Chemotherapy 6, 21. Neff, R. J., Ray, S. A., Benton, W. F. and Wilborn, M. (1964). Induction of synchronous encystment (differentiation) in Acanthamoeba sp, Methods in Cell Physiology 1, 83. Schuster, F. L. (2). Cultivation of pathogenic and opportunistic free-living amebas. Clinical Microbiology Reviews, Schuster, F. L. and Visvesvara, G. S. (4) Free living amoebae as opportunistic and non-opportunistic pathogens of humans and animals. International Journal for Parasitology 34, Schuster, F. L. and Visvesvara, G. S. (1998). Efficacy of novel antimicrobials against clinical isolates of opportunistic amebas. Journal of Eukaryotic Microbiology 4, Seo, S. A., Yong, T. S. and Im, K. (1992). The maintenance of free-living amoebae by cryopreservation. The Korean Journal of Parasitology, 1 3.